Glomerular Capillary Pressure Research Articles

Role of 20-HETE in the impaired myogenic and TGF responses of the Af-Art of Dahl salt-sensitive rats

The present study examined whether 20-HETE production is reduced in the renal vasculature and whether this impairs myogenic or tubuloglomerular feedback (TGF) responses of the afferent arteriole (Af-Art). The production of 20-HETE was 73% lower in renal microvessels of Dahl salt-sensitive rats (SS) rats than in SS.5(BN) rats, in which chromosome 5 from the Brown Norway (BN) rat containing the CYP4A genes was transferred into the SS genetic background. The luminal diameter of the Af-Art decreased by 14.7 ± 1.5% in SS.5(BN) rats when the perfusion pressure was increased from 60 to 120 mmHg, but it remained unaltered in SS rats. Administration of an adenosine type 1 receptor agonist (CCPA, 1 μM) reduced the diameter of the Af-Art in the SS.5(BN) rats by 44 ± 2%, whereas the diameter of the Af-Art of SS rats was unaltered. Autoregulation of renal blood flow (RBF) and glomerular capillary pressure (PGC) was significantly impaired in SS rats but was intact in SS.5(BN) rats. Administration of a 20-HETE synthesis inhibitor, HET0016 (1 μM), completely blocked the myogenic and adenosine responses in the Af-Art and autoregulation of RBF and PGC in SS.5(BN) rats, but it had no effect in SS rats. These data indicate that a deficiency in the formation of 20-HETE in renal microvessels impairs the reactivity of the Af-Art of SS rats and likely contributes to the development of hypertension induced renal injury.

Mechanism of impaired afferent arteriole myogenic response in Dahl salt-sensitive rats: role of 20-HETE.

The afferent arteriole (Af-Art) controls glomerular capillary pressure, an important determinant of glomerular injury. Af-Art myogenic response is mediated by ATP, and ATP signaling is in turn mediated by 20-HETE. Dahl salt-sensitive rats (Dahl SS) have decreased renal 20-HETE production. We hypothesized that Dahl SS have an impaired myogenic response and constrictor response to ATP, due to decreased 20-HETE. Af-Arts from Dahl SS or Dahl salt-resistant rats (Dahl SR) were microdissected and perfused. When myogenic response was induced by increasing Af-Art perfusion pressure from 60 to 140 mmHg, luminal Af-Art diameter decreased in Dahl SR but not in Dahl SS (-3.1 ± 0.8 vs. 0.5 ± 0.8 μm, P < 0.01). The 20-HETE antagonist 20-HEDE (10(-6) M) blocked the myogenic response in Dahl SR but had no effect in Dahl SS. Addition of a subconstrictor concentration of 20-HETE (but not a subconstrictor concentration of norepinephrine) restored the myogenic response in Dahl SS. We then perfused Af-Arts at 60 mmHg and tested the effects of the ATP analog α,β-methylene-ATP (10(-6) M). Maximum ATP-induced constriction was attenuated in Dahl SS compared with Dahl SR (1.5 ± 0.5 vs. 7.4 ± 0.8 μm, P < 0.001). 20-HEDE attenuated ATP-induced Af-Art constriction in Dahl SR but not in Dahl SS, and consequently, ATP-induced constriction was no longer different between strains. In conclusion, Dahl SS have an impaired myogenic response and ATP-induced Af-Art constriction due to a decrease in Af-Art 20-HETE. The impaired myogenic responses may contribute to the nephrosclerosis that develops in Dahl SS.

Aldosterone sensitizes connecting tubule glomerular feedback via the aldosterone receptor GPR30

Increasing Na delivery to epithelial Na channels (ENaC) in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone sensitizes CTGF via a nongenomic mechanism that stimulates CNT ENaC via the aldosterone receptor GPR30. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. During the control period, the concentration of NaCl that elicited a half-maximal response (EC50) was 37.0 ± 2.0 mmol/l; addition of aldosterone 10(-8) mol/l to the CNT lumen caused a left-shift (decrease) in EC50 to 19.3 ± 1.3 mmol/l (P = 0.001 vs. control; n = 6). Neither the transcription inhibitor actinomycin D nor the translation inhibitor cycloheximide prevented the effect of aldosterone (control EC50 = 34.7 ± 1.9 mmol/l; aldosterone+actinomycin D EC50 = 22.6 ± 1.6 mmol/l; P < 0.001 and control EC50 = 32.4 ± 4.3 mmol/l; aldosterone+cycloheximide EC50 = 17.4 ± 3.3 mmol/l; P < 0.001). The aldosterone antagonist eplerenone prevented the sensitization of CTGF by aldosterone (control EC50 = 33.2 ± 1.7 mmol/l; aldosterone+eplerenone EC50 = 33.5 ± 1.3 mmol/l; n = 7). The GPR30 receptor blocker G-36 blocked the sensitization of CTGF by aldosterone (aldosterone EC50 = 16.5 ± 1.9 mmol/l; aldosterone+G-36 EC50 = 29.0 ± 2.1 mmol/l; n = 7; P < 0.001). Finally, we found that the sensitization of CTGF by aldosterone was mediated, at least in part, by the sodium/hydrogen exchanger (NHE). We conclude that aldosterone in the CNT lumen sensitizes CTGF via a nongenomic effect involving GPR30 receptors and NHE. Sensitized CTGF induced by aldosterone may contribute to renal damage by increasing Af-Art dilation and glomerular capillary pressure (glomerular barotrauma).

Mechanistic view of renal protective action of calcium channel blockade.

Calcium channel blockers are one of the most useful antihypertensive agents because of their definite blood pressure lowering action. Although the antihypertensive effect of calcium channel blockers is attributed predominantly to the blockade of L-type calcium channels, recent studies demonstrate that the blockade of other subtypes of calcium channels, including T-type and N-type calcium channels, offers renal protective action because of their beneficial action on glomerular capillary pressure, renal fibrotic process, sympathetic nerve activity and aldosterone synthesis. It requires more extensive studies to clarify whether the ostensibly beneficial actions of these calcium channel blockers are available in a clinical setting.

Angiotensin-converting enzyme 2 mediates hyperfiltration associated with diabetes

The degradation of ANG II by angiotensin-converting enzyme 2 (ACE2), leading to the formation of ANG(1-7), is an important step in the regulation of the renin-angiotensin-aldosterone system (RAAS), and one that is significantly altered in the diabetic kidney. This study examined the role of ACE2 in the hyperfiltration associated with diabetes. Streptozotocin diabetes was induced in male C57BL6 mice and ACE2 knockout (KO) mice. C57BL6 mice were further randomized to receive the selective ACE2 inhibitor MLN-4760. After 2 wk of study, animals were subjected to micropuncture experiments. The renal reserve was further assessed in C57BL6 mice and ACE2 KO mice after exposure to a high-protein diet. The induction of diabetes in wild-type mice was associated with increased renal ACE2 activity, hyperfiltration, and renal hypertrophy. On micropuncture, diabetes was associated with increased tubular free flow and stop-flow pressure, enhanced tubuloglomerular feedback reactivity, and an increased maximal response indicative of increased glomerular hydrostatic capillary pressure. Each of these increases were prevented in diabetic ACE2 KO mice and diabetic mice treated with a selective ACE2 inhibitor for 2 wk. However, unlike chronically treated animals, ACE2 inhibition with MLN-4760 had no acute effect on stop-flow pressure or tubuloglomerular feedback reactivity. ACE2 KO mice also failed to increase their creatinine clearance in response to a high-protein diet. The results of our study suggest that ACE2 plays a key role in the recruitment of the renal reserve and hyperfiltration associated with diabetes.

Rho-kinase Contributes to Pressure-induced Constriction ofRenal Microvessels

Renal afferent arterioles (AFF) regulate glomerular capillary pressure through two main mechanisms: the myogenic response (MYO) and tubuloglomerular feedback (TGF). Because Rho-kinase and nitric oxide synthase (NOS) are established factors that modulate vascular tone, we examined the role of these factors in pressure-induced AFF tone in Wistar-Kyoto rats and in spontaneously hypertensive rats (SHR) using an intravital CCD camera. Elevated renal perfusion pressure elicited marked AFF constriction that was partially inhibited by gadolinium, furosemide and fasudil, which inhibit MYO, TGF and Rho-kinase, respectively; however, this AFF constriction was completely blocked by combined treatment with fasudil+gadolinium or fasudil+furosemide. S-methyl-L-thiocitrulline (SMTC) partially reversed the fasudil-induced inhibition of TGF-mediated, but not that of MYO-mediated, AFF constriction. In SHR, the pressure-induced AFF response was enhanced, and MYO- and TGF-induced constriction were exaggerated. In the presence of gadolinium, SMTC partially mitigated the fasudil-induced inhibition of TGF-mediated AFF constriction. Immunoblot analyses demonstrated that both Rho-kinase activity and neuronal NOS were augmented in SHR kidneys. In conclusion, Rho-kinase contributes to MYO- and TGF-mediated AFF responses, and these responses are enhanced in SHR. Furthermore, neuronal NOS-induced nitric oxide modulates the TGF mechanism. This mechanism constitutes a target for Rho-kinase in TGF-mediated AFF constriction.

Commentary: Microalbuminuria: Past, present and glorious future

In 1969, Keen and colleagues demonstrated increased urinary albumin excretion (microalbuminuria) during a 50-g glucose load in people with diabetes, compared with those with borderline diabetes and controls, applying a sensitive radioimmunoassay method. The 2-h urinary albumin concentration correlated positively with blood glucose and blood pressure in the diabetic group. This led the authors to suggest: ‘The therapeutic implications of this is that treatment of hypertension associated with diabetes may ameliorate or delay the onset of renal disease’. Increased urinary albumin excretion is the net result of glomerular capillary passage and tubular reabsorption. Increased glomerular albumin passage occurs due to functional and structural abnormalities such as: increased glomerular capillary permeability-surface area product (size and charge selectivity); and elevated glomerular hydraulic pressure. Recently, the importance of the glycocalyx for macromolecular transvascular transport has been documented. Poor glycaemic regulation and diabetes reduce the glycocalyx, leading to increased albuminuria. Studies in diabetic animals and man have demonstrated enhanced size and charge selectivity for large macromolecules and raised measured and estimated glomerular capillary hydraulic pressure. The landmark study by Keen et al. created an avalanche of retroand prospective observational studies demonstrating the predictive power of microalbuminuria for: diabetic nephropathy, end-stage renal disease, fatal and nonfatal cardiovascular events in type 1 and type 2 diabetes, hypertension and in the general population. The mechanisms linking microalbuminuria with fatal and nonfatal cardiovascular disease remain poorly understood. Microalbuminuria has been proposed as a marker of widespread endothelial dysfunction that might predispose to enhanced penetrations in the arterial wall of atherogenic lipoprotein particles (the Steno hypothesis). Secondly, microalbuminuria has been suggested simply as a marker of established cardiovascular disease. Thirdly, microalbuminuria is associated with excess of well-known cardiovascular risk factors such as: elevated blood pressure, dyslipoproteinaemia, increased platelet aggregability, insulin resistance and autonomic neuropathy. It is important to recall that no therapy was available for preventing or treating overt diabetic nephropathy in 1969. My own experience between 1975 and 1980 as a senior registrar at Steno Diabetes Center in Copenhagen, caring for approximately 6000 diabetic patients, can be described as a hospital flooded with young type 1 diabetic patients with end-stage renal disease, waiting to die. No therapy was available, including dialysis/transplantation which were not offered by our nephrologists because our patients frequently suffered from several severe vascular and neurological complications. Two to three decades later, numerous studies have demonstrated a renoprotective effect of improved glycaemic regulation, arterial blood pressure reduction, blockade of the renin-angiotensin system independent of blood pressure and intensified multifactorial intervention—with tight glucose regulation, and the use of renin-angiotensin system blockers, aspirin and lipidlowering agents. Glycaemic regulation has mainly a beneficial effect early in the development of diabetic nephropathy i.e. progression from normoalbuminuria to microalbuminuria. Blood pressure reduction, blockade of the renin-angiotensin system and intensified multifactorial intervention act on every stage of development and progression of diabetic nephropathy and end-stage renal disease. It should also be stressed that the above-mentioned treatment modalities can induce regression of urinary albumin excretion rate, and thereby preserve measured glomerular filtration rate. Those studies suggest that microalbuminuria is a risk factor for diabetic nephropathy [end-stage renal disease (ESRD)]. Recently many new biomarkers have been evaluated as predictors for renal risk in diabetic patients, but apart from urinary proteomics (expensive, timeconsuming and with limited access), none has yet Published by Oxford University Press on behalf of the International Epidemiological Association

Abstract 552: Aldosterone Enhances Connecting Tubule Glomerular Feedback (CTGF)

Increasing Na delivery to the connecting tubule (CNT) stimulates epithelial Na channels (ENaC) and dilates the afferent arteriole (Af-Art), a process we call connecting tubule glomerular feedback (CTGF). We hypothesize that aldosterone (aldo) enhances CTGF via a nongenomic mechanism that stimulates CNT ENaC via GPR30 and/or mineralocorticoid receptors (MR). Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing luminal NaCl in the CNT. Addition of aldo 10 -8 M to the CNT potentiated CTGF, seen as a left-shift in the concentration of NaCl that elicited a half-maximal response (EC 50 ), see Figure. The MR blocker eplerenone (10 -5 M) prevented the enhancement of CTGF by aldo (control EC 50 = 32.4 ± 2.3 mM; aldo + eplerenone EC 50 = 35.4 ± 1.7 mM; n = 7). Neither the transcription inhibitor actinomycin D (5x10 -6 M) nor the translation inhibitor cycloheximide (10 -5 M) prevented the effect of aldo (control EC 50 = 33.0 ± 2.0 mM; aldo + actinomycin D EC 50 = 15.4 ± 1.5 mM; n = 6; P &lt; 0.001, control EC 50 = 33.2 ± 2.4 mM; aldo + cycloheximide EC 50 = 11.2 ± 1.3 mM; n = 6; P &lt; 0.001). We conclude that aldo in the lumen of the CNT enhances CTGF via a nongenomic effect possibly involving MR and/or GPR30 receptors. Enhanced CTGF induced by aldosterone may contribute to renal damage by causing increases in Af-Art dilation and glomerular capillary pressure (glomerular barotrauma). Figure. Control CTGF (○) seen as dilation of norepinephrine-preconstricted Af-Arts induced by increasing NaCl in the CNT. Aldo 10 -8 M (•) enhanced CTGF (n = 6; * P &lt; 0.05; ** P &lt; 0.01; *** P &lt; 0.001; vs . control). Vertical dashed lines indicate EC 50 .

Abstract 28: Formation of Podocyte-Derived Microparticles in Diabetic Glomerular Injury

Hypertension is a significant cause of progressive kidney disease, particularly in the presence of diabetes. Under such conditions, increased glomerular capillary pressure subjects podocytes, specialized glomerular epithelial cells critical to filtration, to mechanical stress resulting in podocyte injury/dysfunction. Microparticles (MPs) are small (0.1-1.0 μm), membranous vesicles shed from the cell surface following injury. However, whether podocyte MP formation reflects glomerular injury is unknown. We examined MP formation by podocytes in vitro and in vivo. Conditionally immortalized human podocytes were exposed to 10% equibiaxial cyclic stretch (a mimic of increased intraglomerular pressure), high glucose (HG, 25 mM), mannitol (osmotic control), angiotensin II (Ang II, 500 nM) or transforming growth factor beta (TGF-β, 5 ng/mL). Additionally, urinary podocyte MPs were quantified in two mouse models of diabetic kidney disease: streptozotocin (STZ) and OVE26. MPs were characterized by nanoparticle tracking analysis and quantified by Annexin V (total MPs) or podocalyxin (podocyte MPs) labeling and flow cytometry. Podocyte-derived vesicles were identifiable in both media and urine samples with a mean size of 236 nm by nanoparticle tracking analysis. In vitro, cyclic stretch was associated with a 3-fold increase in MP release after 24 hours (P&lt;0.01, n=6). HG increased MP release 5-fold after 24 hours (P&lt;0.05, n=6). Mannitol had no effect on MP formation by either normal or stretched podocytes and neither Ang II, nor TGF-β altered podocyte MP formation over 24 hours. In vivo, both models of diabetes displayed typical hallmarks of renal injury (proteinuria, mesangial expansion). In OVE26 mice urinary podocyte MPs were elevated compared with their wild-type littermates (17479±8329 vs. 7 ±7, P&lt;0.05, n=5-7). Similarly, STZ-treated mice displayed increased urinary podocyte MPs as compared with untreated (18035±3813 vs. 43±34, P&lt;0.001, n=9-18) and urinary MPs levels were positively correlated with albuminuria (r2=0.74, P&lt;0.01). Our results suggest that podocytes produce MPs which are released into urine and are indicative of glomerular injury. Such processes may be mediated by intraglomerular capillary pressure and hyperglycemia.

Connecting Tubule Glomerular Feedback in Hypertension

In Dahl salt-sensitive rats (Dahl SS), glomerular capillary pressure increases in response to high salt intake and this is accompanied by significant glomerular injury compared with spontaneously hypertensive rats with similar blood pressure. Glomerular capillary pressure is controlled mainly by afferent arteriolar resistance, which is regulated by the vasoconstrictor tubule glomerular feedback (TGF) and the vasodilator connecting TGF (CTGF). We hypothesized that Dahl SS have a decreased TGF response and enhanced TGF resetting compared with spontaneously hypertensive rats, and that these differences are attributable in part to an increase in CTGF. In vivo, using micropuncture we measured stop-flow pressure (a surrogate of glomerular capillary pressure). TGF was calculated as the maximal decrease in stop-flow pressure caused by increasing nephron perfusion, TGF resetting as the attenuation in TGF induced by high salt diet, and CTGF as the difference in TGF response before and during CTGF inhibition with benzamil. Compared with spontaneously hypertensive rats, Dahl SS had (1) lower TGF responses in normal (6.6±0.1 versus 11.0±0.2 mm Hg; P<0.001) and high-salt diets (3.3±0.1 versus 10.1±0.3 mm Hg; P<0.001), (2) greater TGF resetting (3.3±0.1 versus 1.0±0.3 mm Hg; P<0.001), and (3) greater CTGF (3.4±0.4 versus 1.2±0.1 mm Hg; P<0.001). We conclude that Dahl SS have lower TGF and greater CTGF than spontaneously hypertensive rats, and that CTGF antagonizes TGF. Furthermore, CTGF is enhanced by a high-salt diet and contributes significantly to TGF resetting. Our findings may explain in part the increase in vasodilatation, glomerular capillary pressure, and glomerular damage in SS hypertension during high salt intake.

Synthetic oleanane triterpenoids: magic bullets or not?

A recent clinical trial in patients with chronic kidney disease (CKD) and diabetic nephropathy demonstrated that bardoxolone methyl (CDDO-ME) increases estimated glomerular filtration rate (eGFR) by an unknown mechanism. The paper by Ding et al. suggests that short-term administration of a CDDO-ME analog increases GFR by increasing glomerular surface area. However, changes in other renal hemodynamic parameters cannot be excluded. Vigorous testing of CDDO-ME and highly purified analogs is warranted to determine their physiology, pharmacology, and efficacy and to exclude serious side effects.

Pleiotropic effects of new generation calcium channel blockers on cardiovascular protections

Calcium channel blockers (CCBs) are widely used to treat hypertension and generally act on L-type calcium channels. Recent studies have reported that some CCBs can also block channels belonging to other subtypes, such as N-type and T type channels. N-type calcium channels are present at sympathetic nerve terminals. T-type calcium channels are identified as an important molecular target in various organs such as the cardiac sinus node and the afferent and efferent arterioles. Blockade of N-type or T-type calcium channels resulted in reduction of glomerular capillary pressure, a stabilization of renin-angiotensin-aldosterone system and sympathetic nervous system. Therefore, the blockade of non-L-type calcium channels has unique pharmacological features of renoprotective, vascular endothelial protective, and cardioprotective effects. These new generation CCBs have special therapeutic qualities beyond their primary blood pressure lowering effects through the blocking of L-type calcium channels. The strategy for hypertension treatment with CCBs has entered a new era. We review the pleiotropic effects of CCBs on cardiovascular protections.

Abstract 490: Constrictor Mechanism of the Afferent Arteriole Initiated by Na/H Exchanger in the Nephron

The afferent arteriole (Af-Art) accounts for most of renal vasculature resistance, thus controlling glomerular filtration rate and renal function. Two mechanisms have been described in which the nephron helps control Af-Art resistance, namely tubuloglomerular feedback (TGF), and connecting tubule glomerular feedback (CTGF). TGF is a constrictor mechanism initiated at the macula densa by the Na/K/2Cl cotransporter (NKCC2), while CTGF is a dilator mechanism initiated at the connecting tubule by the epithelial Na channel (ENaC). However, when NKCC2 is blocked by furosemide, CTGF-induced Af-Art dilation is not evident, thus we hypothesize that there is a constrictor mechanism that is furosemide-insensitive and counters CTGF. To test this, we used in vivo micropuncture of single nephrons. Stop-flow pressure (P SF ) was measured as an index of glomerular capillary pressure (which decreases when the Af-Art constricts). Two consecutive P SF curves were generated by raising nephron perfusion from 0 to 40 nL/min while adding drugs to the tubular perfusate. The decrease in P SF induced by increasing nephron perfusion was blocked by furosemide 10 -4 M (control: 7.9±0.2 mmHg, furosemide: 0.4±0.2 mmHg, P &lt;0.001, n=6), but was partially restored when blocking CTGF with the ENaC inhibitor benzamil 10 -6 M (furosemide: 0.2±0.1 mmHg, furosemide+benzamil: 4.3±0.3 mmHg, P &lt;0.001, n=6). A possible explanation for these observations is that TGF may be activated to some degree by Na/H exchanger (NHE)-mediated Na entry at the macula densa when NKCC2 is blocked. When we added the NHE blocker dimethylamiloride (DMA, 10 -4 M) in the presence of furosemide and benzamil, the decrease in PSF was significantly prevented (furosemide+benzamil: 4.6±0.3 mmHg, furosemide+benzamil+DMA: 1.1±0.2 mmHg, P &lt;0.001, n=6). Therefore, we conclude that a constrictor mechanism of the Af-Art is initiated by NHE in the nephron and is observable when NKCC2-mediated TGF and ENaC-mediated CTGF have been blocked.

The Challenge and Response of Podocytes to Glomerular Hypertension

Glomerular hypertension (ie, increased glomerular capillary pressure), has been shown to cause podocyte damage progressing to glomerulosclerosis in animal models. Increased glomerular capillary pressure results in an increase in wall tension that acts primarily as circumferential tensile stress on the capillary wall. The elastic properties of the glomerular basement membrane (GBM) and the elastic as well as contractile properties of the cytoskeleton of the endothelium and of podocyte foot processes resist circumferential tensile stress. Whether the contractile forces generated by podocytes are able to equal circumferential tensile stress to effectively counteract wall tension is an open question. Mechanical stress is transmitted from the GBM to the actin cytoskeleton of podocyte foot processes via cell-matrix contacts that contain mainly integrin α3β1 and a variety of linker, scaffolding, and signaling proteins, which are not well characterized in podocytes. We know from in vitro studies that podocytes are sensitive to stretch, however, the crucial mechanosensor in podocytes remains unclear. On the other hand, in vitro studies have shown that in stretched podocytes specific signaling cascades are activated, the synthesis and secretion of various hormones and their receptors are increased, cell-cycle arrest is reinforced, cell adhesion is altered through secretion of matricellular proteins and changes in integrin expression, and the actin cytoskeleton is reorganized in a way that stress fibers are lost. In summary, current evidence suggests that in glomerular hypertension podocytes primarily aim to maintain the delicate architecture of interdigitating foot processes in the face of an expanding GBM area.

Role of 20-HETE in the antihypertensive effect of transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats

This study examined whether substitution of chromosome 5 containing the CYP4A genes from Brown Norway rat onto the Dahl S salt-sensitive (SS) genetic background upregulates the renal production of 20-HETE and attenuates the development of hypertension. The expression of CYP4A protein and the production of 20-HETE were significantly higher in the renal cortex and outer medulla of SS.5(BN) (chromosome 5-substituted Brown Norway rat) consomic rats fed either a low-salt (LS) or high-salt (HS) diet than that seen in SS rats. The increase in the renal production of 20-HETE in SS.5(BN) rats was associated with elevated expression of CYP4A2 mRNA. MAP measured by telemetry rose from 117 ± 1 to 183 ± 5 mmHg in SS rats fed a HS diet for 21 days, but only increased to 151 ± 5 mmHg in SS.5(BN) rats. The pressure-natriuretic and diuretic responses were twofold higher in SS.5(BN) rats compared with SS rats. Protein excretion rose to 354 ± 17 mg/day in SS rats fed a HS diet for 21 days compared with 205 ± 13 mg/day in the SS.5(BN) rats, and the degree of glomerular injury was reduced. Baseline glomerular capillary pressure (Pgc) was similar in SS.5(BN) rats (43 ± 1 mmHg) and Dahl S (44 ± 2 mmHg) rats. However, Pgc increased to 59 ± 3 mmHg in SS rats fed a HS diet for 7 days, while it remained unaltered in SS.5(BN) rats (43 ± 2 mmHg). Chronic administration of an inhibitor of the synthesis of 20-HETE (HET0016, 10 mg·kg(-1)·day(-1) iv) reversed the antihypertensive phenotype seen in the SS.5(BN) rats. These findings indicate that the transfer of chromosome 5 from the BN rat onto the SS genetic background increases the renal expression of CYP4A protein and the production of 20-HETE and that 20-HETE contributes to the antihypertensive and renoprotective effects seen in the SS.5(BN) consomic strain.

Glomerular hyperfiltration: a marker of early renal damage in pre-diabetes and pre-hypertension

Glomerular hyperfiltration is a characteristic functional abnormality in insulin-dependent diabetes mellitus and occurs in the large majority of young Type 1 diabetic patients. Hyperfiltration is hypothesized to be a precursor of intraglomerular hypertension leading to albuminuria. Glomerular filtration rate (GFR) then falls progressively in parallel with a further rise in albuminuria which may lead, in the long run, to end-stage renal failure. Experimental and clinical studies have shown that glomerular hyperfiltration can occur also in hypertension. However, whether hyperfiltration occurs also in early stages of hyperglycaemia and high blood pressure, such as in pre-diabetes and pre-hypertension, is not well known. In this issue of the Journal, Okada and Coll studied a large Japanese population showing that in pre-diabetic and pre-hypertensive subjects, the prevalence of hyperfiltration is proportional to the level of glucose and blood pressure, respectively. One main problem with the diagnosis of hyperfiltration is that no generally accepted definition is available due to the strong inverse correlation of GFR with age. To overcome this limitation, Okada et al. calculated the upper normal limit of GFR in a large healthy Japanese sample divided into 10-year age groups, providing ageand sex-specific reference values. For hyperfiltration to develop, the concomitant action of a variety of pathogenetic factors are needed including increased body mass index, hyperinsulinaemia, activation of the sympathetic nervous system, hyperleptinaemia, increased oxidative stress, inflammatory cytokines, etc. Another mechanism accounting for increased GFR in obese persons is increased NaCl intake. In addition, a significant role of increased Na reabsorption in the pathophysiology of glomerular hyperfiltration in obesity and hypertension has been described. Pharmacological agents with action on the renin–angiotensin system are effective in reducing glomerular hypertension which accounts for their efficacy in preventing progression of microalbuminuria in diabetes and hypertension. According to current guidelines, only low GFR and microalbuminuria or proteinuria should be considered as markers of renal dysfunction. Due to the strong association between hyperfiltration and risk of microalbuminuria found in diabetes and hypertension, hyperfiltration should be regarded as a precursor of nephropathy in these clinical conditions. More extensive use of markers of early organ damage may help clinicians to reach a more timely decision about the initiation of treatment and thus delay cardiovascular complications. Hyperglycaemia and high blood pressure in people with hyperfiltration should be treated earlier to prevent the progression of renal dysfunction to chronic kidney disease. Increased GFR, also called hyperfiltration, is a proposed mechanism for renal injury in several clinical conditions. According to the Brenner theory [1], low nephron number at birth explains why some individuals are prone to developing progressive renal damage later in life when other risk factors become operative. At the single-nephron level, hyperfiltration is hypothesized to be a precursor of intraglomerular hypertension leading to albuminuria. Increased glomerular capillary hydraulic pressure may be due to changes in systemic arterial pressure and/or changes in efferent and afferent arteriolar resistances. In the absence of therapeutic interventions, GFR then falls progressively in parallel with a further rise in albuminuria which may lead, in the long run, to end-stage renal failure (Figure 1). Human models of renal injury are in keeping with this pathogenetic view. Glomerular hyperfiltration has been observed in patients with unilateral renal agenesis [2], congenitally reduced nephron number [3] and acquired reduction in renal mass [4]. These individuals are prone to developing proteinuria early in life in association with glomerular sclerosis. It is suggested that this model of renal injury may apply to the early diabetic or hypertensive kidney.

Temporal characterization of the development of renal injury in FHH rats and FHH.1BNcongenic strains

The present study examined the effect of transfer of portions of chromosome 1 that includes (FHH.1(BN) AR(+) strain) or excludes (control FHH.1(BN) AR(-) strain) a 4.3-Mb region from the Brown Norway (BN) rat that restores the autoregulation (AR) of renal blood flow (RBF) on the development of hypertension and renal injury in congenic strains of Fawn Hooded Hypertensive (FHH) rats. FHH and control AR(-) rats exhibited poor autoregulation of RBF, and glomerular capillary pressure (Pgc) rose by 19 ± 2 mmHg in FHH rats when renal perfusion pressure (RPP) was increased from 100 to 150 mmHg. In contrast, RBF was well autoregulated in the AR(+) strain, and Pgc only increased by 3 ± 1 mmHg when RPP was increased over this range. Baseline mean arterial pressure (MAP) at 12 wk of age was similar in all strains and averaged 122 mmHg. MAP increased significantly in FHH rats and was significantly higher by 12 mmHg in 21-wk-old FHH rats than in the FHH.1(BN) congenic strains. Protein excretion rose from 5 ± 1 to 397 ± 29 mg/day in 6- vs. 21-wk-old FHH rats. In contrast, protein excretion only increased to 139 ± 21 mg/day in the control AR(-) strain, and it did not increase significantly in the AR(+) strain. Glomerular permeability to albumin was similar in all strains at 6 wk of age. It increased significantly in 9-wk-old FHH and control AR(-) rats, but not in the AR(+) strain. The levels of matrix metalloproteinase (MMP)-2 and transforming growth factor (TGF)-β2 protein were significantly higher in the renal cortex of 9-wk-old FHH rats compared with the levels seen in the AR(+) strain. These data indicate that transfer of a 4.3-Mb region of BN chromosome 1 into the FHH genetic background improves autoregulation of RBF, normalizes Pgc, and slows the progression of renal disease.

Injury, failure or success? Renin-angiotensin system inhibition in acute kidney injury.

Sir, The use of inhibitors of the renin–angiotensin system (RAS) in kidney disease requires that a balance be struck between long-term benefits to kidney health on the one hand and reductions in excretory function on the other. In proteinuric chronic kidney disease (CKD) management, some degree of functional reduction following RAS inhibition is generally considered acceptable in anticipation of longer-term preservation of kidney health. In some settings, such as pre-dialysis, it may be more important to seek maximization of excretory function and avoid RAS inhibition [1]. When it comes to acute kidney injury (AKI), RAS inhibition is perceived to be a predisposing factor. It should be noted, however, that the classification systems for AKI remain based on a reduction in excretory ‘function’ rather than measures of kidney ‘damage’. In the case of RAS inhibition, this is more than a semantic difference. By preferentially vasodilating the efferent arteriole and removing the angiotensin II-mediated maintenance of glomerular pressure, RAS inhibition will naturally reduce excretory function in settings where intrarenal perfusion pressure is compromised. However, such a reduction in excretory function does not necessarily entail any greater injury. Lower glomerular capillary pressures have not been linked to any particular pathological lesion. Furthermore, by vasodilating the efferent arteriole, RAS inhibition may improve perfusion of the peritubular capillaries which lie downstream of the glomerular circulation. A reduction in glomerular filtration rate reduces the reabsorptive workload of the tubular cells, the common victims in ischaemic AKI. Lower excretory function consequent upon the intrarenal effects of RAS inhibition may thus be protective for tubular cells, an extension of the concept of ‘acute renal success’ [2]. Consistent with this, RAS inhibition was renoprotective in a number of animal models of AKI, including ischaemia [3]. Therefore, theoretically, the natural history of AKI occurring on a background of RAS inhibition thus might manifest a tendency to present greater dysfunction but more rapid/complete ultimate renal recovery relative to this initial degree of dysfunction. That said, we are of the view that discontinuation of RAS inhibition in the setting of threatened renal perfusion is mandatory to maximize excretory function, prevent incipient ATN at the pre-renal stage, and minimize the risk of a requirement for dialysis which itself carries a significant morbidity burden. There may nevertheless be a population of patients with AKI for whom RAS inhibition is actually beneficial. When patients have already commenced dialysis for AKI and the systemic perfusion pressure is maintained/elevated, a short-term reduction in excretory function following RAS inhibition is potentially of little consequence and could be associated with longer-term benefits to renal health. Since RAS inhibitors are anti-fibrotic (via reduction of transforming growth factor-beta signalling [4]), their prompt commencement in patients with diverse kidney injuries may help prevent scarring. Tubuloprotective effects theoretically could be of benefit in glomerular pathologies compromising downstream peritubular capillary perfusion (for example vasculitis and thrombotic microangiopathies) as well as in tubular disorders (such as acute tubular necrosis). A trial of RAS inhibition in carefully selected patients with AKI requiring dialysis would not be unethical since (i) there is a theoretical basis for benefit, (ii) animal studies have been consistent with benefit and (iii) RAS inhibitors are already advocated for the preservation of residual renal function in patients established on dialysis. Inclusion criteria for such a study would specifically need to avoid recruitment of patients at risk of systemic hypotension upon RAS inhibition. Meaningful primary end points would be recovery of independent renal function or the degree of functional recovery. Acute functional changes are a poor outcome measure for the reasons discussed. Previous observational studies have, however, used this end point in assessing the renal effect of RAS inhibition in settings such as radiocontrast administration or surgery [5]. Better evidence to tailor the appropriate use of RAS inhibition in both acute and chronic kidney disease should be of benefit to the large numbers of patients who may derive benefit, or harm, from their use. Conflict of interest statement. None declared.

P.2.b.023 Rodent magnesium-deficiency models for depression and their relationship to NMDA receptor/Nitric Oxide pathways

Colocalization of demethylating enzymes and NOS and functional effects of methylarginines in rat kidney. NG-monomethylarginine (L-NMA) and asymmetric NG, NG-dimethylarginines (ADMA) are endogenous inhibitors of cellular L-arginine uptake and/or nitric oxide (NO) synthesis that are implicated in renal parenchymal and Dahl salt-sensitive hypertension. Since the L-arginine:(L-NMA+ADMA) ratio determines NO synthase (NOS) activity, we compared the immunohistochemical distribution of NOS with NG, NG-dimethylarginine dimethylaminohydrolase (DDAH), which inactivates dimethylarginines (DMA) and L-NMA by hydrolysis to L-citrulline. Neuronal NOS (nNOS) was expressed predominantly in tubular epithelial cells of macula densa (MD), endothelial NOS (eNOS) in vascular endothelial cells (EC), and inducible NOS (iNOS) quite widely in tubular epithelium, including proximal tubules (PT), thick ascending limbs of Henle (TAL), distal convoluted tubule and intercalated cells (IC) of the collecting duct. Immunostaining for DDAH was present in PT, TAL, MD, and IC, and was also present in the glomerulus, Bowman's capsule, and endothelium of blood vessels. DDAH was detected in small vesicles of TAL and PT by electron microscopic (EM) immunocytochemistry. To study the effects of methylarginines on tubuloglomerular feedback (TGF) response, vehicle or methylarginines (10−3 M) were added to artificial tubular fluid (ATF) perfused orthogradely from the late PT at 40 nl. min−1 while assessing changes in glomerular capillary pressure from proximal stop flow pressure (PSF). Whereas the maximal TGF responses were unchanged by vehicle (ΔTGF 0 ± 0%) or symmetric DMA (SDMA; +1 ± 2%, NS), they were enhanced by L-NMA (+22 ± 4%, P < 0.001) and asymmetric DMA (ADMA; +28 ± 3%, P < 0.001). Since L-arginine transport can regulate renal epithelial NO generation, methylarginines (10−3 M) or vehicle were co-perfused orthogradely with [3H]-L-arginine from the late PT and collected at the early distal tubule to study arginine uptake from the perfused loop of Henle. All methylarginines reduced fractional loop [3H] absorption significantly (P < 0.001; vehicle, 84 ± 6; ADMA, 49 ± 6; SDMA, 56 ± 6; L-NMA, 41 ± 6%). In conclusion, sites of DDAH expression in the vasculature or nephron are all sites of expression of an isoform of NOS. L-NMA, ADMA, and SDMA all inhibit renal tubular L-arginine uptake, whereas L-NMA and ADMA, but not SDMA, enhance TGF responses. Therefore, DDAH may regulate the cellular L-arginine: methylarginine levels in specific renal cells, thereby governing cell-specific L-arginine uptake and NO generation in renal tubular epithelium.

A Maladaptive Role for EP4 Receptors in Podocytes

Inhibition of p38 mitogen-activated protein kinase and cyclooxygenase-2 reduces albuminuria in models of chronic kidney disease marked by podocyte injury. Previously, we identified a feedback loop in podocytes whereby an in vitro surrogate for glomerular capillary pressure (i.e., mechanical stretch) along with prostaglandin E(2) stimulation of its EP4 receptor induced cyclooxygenase-2 in a p38-dependent manner. Here we asked whether stimulation of EP4 receptors would exacerbate glomerulopathies associated with enhanced glomerular capillary pressure. We generated mice with either podocyte-specific overexpression or depletion of the EP4 receptor (EP4(pod+) and EP4(pod-/-), respectively). Glomerular prostaglandin E(2)-stimulated cAMP levels were eightfold greater for EP4(pod+) mice compared with nontransgenic (non-TG) mice. In contrast, EP4 mRNA levels were >50% lower, and prostaglandin E(2)-induced cAMP synthesis was absent in podocytes isolated from EP4(pod-/-) mice. Non-TG and EP4(pod+) mice underwent 5/6 nephrectomy and exhibited similar increases in systolic BP (+25 mmHg) by 4 weeks compared with sham-operated controls. Two weeks after nephrectomy, the albumin-creatinine ratio of EP4(pod+) mice (3438 μg/mg) was significantly higher than that of non-TG mice (773 μg/mg; P < 0.0001). Consistent with more severe renal injury, the survival rate for nephrectomized EP4(pod+) mice was significantly lower than that for non-TG mice (14 versus 67%). In contrast, 6 weeks after nephrectomy, the albumin-creatinine ratio of EP4(pod-/-) mice (753 μg/mg) was significantly lower than that of non-TG mice (2516 μg/mg; P < 0.05). These findings suggest that prostaglandin E(2), acting via EP4 receptors contributes to podocyte injury and compromises the glomerular filtration barrier.